Mutants which produce a potentiator of bacillus pesticidal activity

ABSTRACT

This invention is directed to Bacillus mutants which produce a factor which potentiates the pesticidal activity of a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties.

[0001] This application is a continuation-in-part application ofapplication Ser. No. 08/146,852, filed Nov. 3, 1993, which is acontinuation-in-part of application Ser. No. 08/095,240, filed Jul. 20,1993, which is a continuation-in-part of applcation Ser. No. 07/990,202,filed Dec. 14, 1992, which is a continuation-in-part of application Ser.No. 07/971,786, filed Nov. 5, 1992.

1. FIELD OF THE INVENTION

[0002] The invention is related to a mutant Bacillus strain whichproduces a factor which potentiates the pesticidal activity of aBacillus related pesticide, a chemical pesticide and/or a virus withpesticidal properties, in which such a factor is obtained in largeramounts or has a greater potentiating activity compared to the parentalstrain, and methods for producing such mutant strains. The inventionalso relates to methods for obtaining the factor.

2. BACKGROUND OF THE INVENTION

[0003] Every year, pests detrimental to agriculture, forestry, andpublic health cause losses in the millions of dollars. Variousstrategies have been used to control such pests.

[0004] One strategy is the use of chemical pesticides with a broad rangeor spectrum of activity. However, there are a number of disadvantageswith using chemical pesticides. Specifically, because of their broadspectrum of activity, these pesticides may destroy non-target organismssuch as beneficial insects and parasites of destructive pests.Additionally, chemical pesticides are frequently toxic to animals andhumans. Furthermore, targeted pests frequently develop resistance whenrepeatedly exposed to such substances.

[0005] Another strategy involves the use of biopesticides to controlinsect, fungal and weed infestations. Biopesticides are naturallyoccurring pathogens and/or the substances produced by these pathogens.The advantage of using biopesticides is that they are generally lessharmful to non-target organisms and the environment as a whole comparedto chemical pesticides.

[0006] 2.1. Bacillus thurinciensis

[0007] The most widely used biopesticide is Bacillus thuringiensis.Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacteriumthat is widely distributed in nature, especially in soil and insect-richenvironments. During sporulation, Bacillus thuringiensis produces aparasporal crystal inclusion(s) which is insecticidal upon ingestion tosusceptible insect larvae of the orders Lepidoptera, Diptera, andColeoptera. The inclusions may vary in shape, number, and composition.They are comprised of one or more proteins called delta-endotoxins,which may range in size from 27-140 kDa. The insecticidaldelta-endotoxins are generally converted by proteases in the larval gutinto smaller (truncated) toxic polypeptides, causing midgut destruction,and ultimately, death of the insect (Höfte and Whiteley, 1989,Microbiological Reviews 53:242-255).

[0008] There are several Bacillus thuringiensis strains that are widelyused as biopesticides in the forestry, agricultural, and public healthareas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensissubsp. aizawai produce delta-endotoxins specific for Lepidoptera. Adelta-endotoxin specific for Coleoptera is produced by Bacillusthuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No.4,766,203). Furthermore, Bacillus thuringiensis subsp. israelensisproduces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S.Pat. No. 4,166,112).

[0009] Other Bacillus thuringiensis strains specific for dipteran pestshave also been described. A Bacillus thuringiensis isolate has beendisclosed which is toxic to Diptera and Lepidoptera (Hodgman et al.,1993, FEMS Microbiology Letters 114:17-22). SDS polyacrylamide gelelectrophoresis of the purified crystal delta-endotoxin from thisisolate revealed three protein species which are related to CryIA(b),CryIB, and CryIIA toxins. There has also been disclosed a Bacillusthuringiensis isolate which produces a dipteran-active crystal comprisedof proteins with molecular weights of 140, 122, 76, 72, and 38 kDa(Payne, 1994, U.S. Pat. No. 5,275,815). EPO 480,762 discloses five B.t.strains which are each active against dipteran pests; each also have aunique crystal delta-endotoxin pattern.

[0010] Several Bacillus thuringiensis strains have been described whichhave pesticidal activity against pests other then Lepidoptera,Coleoptera, and Diptera. Five Bacillus thuringiensis strains have beendisclosed which produce delta-endotoxins that are toxic againstnematodes (Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303426 Bi). There has also been disclosed a Bacillus tiuringiensis strain,PS81F, which can be used to treat humans and animals hosting parasiticprotozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799A2). Several Bacillus thuringiensis isolates have also been disclosedwith activity against acaride pests. These isolates produce crystalscomprised of proteins with molecular weights in the (wide) range of 35kDa to 155 kDa (Payne, Cannon, and Bagley, 1992, PCT Application No. WO92/19106). There have also been disclosed Bacillus thuringiensis strainswith activity against pests of the order Hymenoptera (Payne, Kennedy,Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); withactivity against pests of the order Hemiptera (Payne and Cannon, 1993,U.S. Pat. No. 5,262,159); with activity against fluke pests (Hickle,Sick, Schwab, Nara, and Payne, 1993, U.S. Pat. No. 5,262,399; and withactivity against pests of the order Phthiraptera (Payne and Hickle, 993,U.S. Pat. No. 5,273,746). Furthermore, another strain of Bacillusthuringiensis subsp, kurstaki, WB3S-16, isolated from Australian sheepwool clippings, has been disclosed that is toxic to the biting louseDamalinia ovis, a Phthiraptera pest (Drummond, Miller, and Pinnock,1992, J. Invert. Path. 60:102-103).

[0011] The delta-endotoxins are encoded by cry (crystal protein) geneswhich are generally located on plasmids. The cry genes have been dividedinto six classes and several subclasses based on relative amino acidhomology and pesticidal specificity. The major classes areLepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII);Coleoptera-specific (cryIII); Diptera-specific (cryIV) (Höfte andWhiteley, 1989, Microbiological Reviews 53:242-255); Coleoptera- andLepidoptera-specific (referred to as cryV genes by Tailor et al., 1992,Molecular Microbiology 6:1211-1217); and Nematode-specific (referred toas cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology10:271-275).

[0012] Delta-endotoxins have been produced by recombinant DNA methods.The delta-endotoxins produced by recombinant DNA methods may or may notbe in crystal form.

[0013] Some strains of Bacillus thuringiensis have been shown to producea heat-stable pesticidal adenine-nucleotide analog, known as β-exotoxintype I or thuringiensin, which is pesticidal alone (Sebesta et al., inH. D. Burges (ed.), Microbial Control of Pests and Plant Diseases,Academic Press, New York, 1980, pp. 249-281). β-exotoxin type I has beenfound in the supernatant of some Bacillus thuringiensis cultures. It hasa molecular weight of 701 and is comprised of adenosine, glucose, andallaric acid (Farkas et al., 1977, Coll. Czechosslovak Chem. Comm.42:909-929; Lüthy et al., in Kurstak (ed.), Microbial and ViralPesticides, Marcel Dekker, N.Y., 1982, pp. 35-72). Its host rangeincludes, but is not limited to, Musca domestica, Mamestra configurataWalker, Tetranychus urticae, Drosophila melanogaster, and Tetranychuscinnabarinus. The toxicity of β-exotoxin type I is thought to be due toinhibition of DNA-directed RNA polymerase by competition with ATP. Ithas been shown that β-exotoxin type I is encoded by a cry plasmid infive Bacillus thuringiensis strains (Levinson et al., 1990, J.Bacteriol. 172:3172-3179). β-exotoxin type I was found to be produced byBacillus thuringiensis subsp. thuringiensis serotype 1, Bacillusthuringiensis subsp. tolworthi serotype 9, and Bacillus thuringiensissubsp. darmstadiensis serotype 10.

[0014] Another β-exotoxin classified as β-exotoxin type II has beendescribed (Levinson et al., 1990, J. Bacteriol. 172:3172-3179).β-exotoxin type II was found to be produced by Bacillus thuringiensissubsp. morrisoni serotype 8ab and is active against Leptinotarsadecemlineata. The structure of β-exotoxin type II is not completelyknown, but is significantly different from that of β-exotoxin type I inthat a pseudouridine moiety is in the place of adenine in whichattachment to the ribose ring is at a position that would otherwise beoccupied by a proton (Levinson, in Hickle and Finch (eds.), AnalyticalChemistry of Bacillus thuringiensis, ACS Symposium Series, Washington,D.C., 1990, pp. 114-136). Furthermore, there is only one signal in theproton NMR spectrum corresponding to the nucleoside base (at 7.95 ppm),and does not have a ribose-type anomeric protein signal (5.78 ppm).

[0015] Other water soluble substances that have been isolated fromBacillus thuringiensis include alpha-exotoxin which is toxic against thelarvae of Musca domestics (Luthy, 1980, FEMS Microbiol. Lett. 8:1-7);gamma-exotoxins, which are various enzymes including lecithinases,chitinases, and proteases, the toxic effects of which are expressed onlyin combination with beta-exotoxin or delta-endotoxin (Forsberg et al.,1976, Bacillus thuringiensis: Its Effects on Environmental Quality,National Research Council of Canada, NRC Associate Committee onScientific Criteria for Environmental Quality, Subcomittees onPesticides and Related Compounds and Biological Phenomena); sigmaexotoxin which has a structure similar to beta-exotoxin, and is alsoactive against Leptinotarsa decemlineata (Argauer et al., 1991, J.Entomol. Sci. 26:206-213); and anhydrothuringiensin (Prystas et al.,1975, Coll. Czechosslovak Chem. Comm. 40:1775).

[0016] 2.2. Zwittermicin

[0017] A substance has been isolated from Bacillus cereus which inhibitsthe growth of the plant pathogen Phytophthora medicaginis and reducesthe infection of alfalfa (see, for example, U.S. Pat. Nos. 4,877,738 and4,878,936). No other activity was disclosed. The following structure hasbeen elucidated for zwittermicin A (He et al., Tet. Lett. 35:2499-2502):

3. OBJECTS OF THE INVENTION

[0018] The art has strived to achieve increased mortality of B.t.formulations. Means have included searching for new strains withincreased mortality, attempting to engineer present strains, andattempting to design more effective formulations by combining B.t.spores and crystals with new pesticidal carriers chemical pesticides, orenhancers (see, for example, U.S. Pat. No. 5,250,515, a trypsininhibitor). It is therefore an object of the present invention topotentiate the pesticidal activity of pesticides. It is also an objectof the invention to isolate strains that produce large amounts ofpotentiator.

4. SUMMARY OF THE INVENTION

[0019] The invention relates to a mutant Bacillus strain which producesa factor which potentiates the pesticidal activity of a Bacillus relatedpesticide, wherein the amount of the factor produced by the mutant isgreater than the amount of the factor produced by a correspondingparental strain. In a specific embodiment, the Bacillus strain isselected from the group consisting of Bacillus subtilus, Baciiluslicheniformis, and Bacillus thuringiensis.

[0020] The factor produced by said mutant is a potentiator. As definedherein, a “potentiator” is a substance which has no significantpesticidal activity, e.g., having an LC₅₀ (LC₅₀ is the concentration ofthe substance required to kill 50% of the pests) of more than about 3000μg/g as assayed by bioassay (see Section 6) but-acts to increase thepesticidal activity of a Bacillus related pesticide at least about 50%and does not cause larval stunting. As noted in Section 2, othersubstances capable of enhancing pesticidal activity known in the artsuch as delta-endotoxins, trypsin inhibitors and exotoxins havepesticidal activity.

[0021] In a specific embodiment, the factor is water soluble. As definedherein, a substance or compound is “water soluble” if at least about 1mg of a substance can be dissolved in 1 ml of water. The factor may alsopotentiate the pesticidal activity of a chemical pesticide and/or avirus with pesticidal properties.

[0022] As defined herein, “a Bacillus related pesticide” is a Bacillus(e.g., Bacillus thuringiensis or Bacillus subtilis) strain, spore, orsubstance, e.g., protein or fragment thereof having activity against orwhich kill pests or a microorganism capable of expressing a Bacillusgene encoding a Bacillus protein or fragment thereof having activityagainst or which kill pests (e.g., Bacillus thuringiensisdelta-endotoxin) and an acceptable carrier (see Section 5.2., infra, forexamples of such carriers). The pest may be, for example, an insect, anematode, a mite, or a snail. A microorganism capable of expressing aBacillus gene encoding a Bacillus protein or fragment thereof havingactivity against or which kill pests inhabits the phylloplane (thesurface of the plant leaves), and/or the rhizosphere (the soilsurrounding plant roots), and/or aquatic environments, and is capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms and provide for thestable maintenance and expression of a Bacillus gene encoding a Bacillusprotein or fragment thereof having activity against or which kill pests.Examples of such microorganisms include but are not limited to bacteria,e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella,Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, Alcaligenes, and Clostridium; algae, e.g., familiesCyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae,Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae,Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae,Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g.,genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces,Rhodotorula, and Aureobasidium.

[0023] As defined herein, “pesticidal activity” measures the amount ofactivity against a pest through killing or stunting of the growth of thepest or protecting the plant from pest infestation.

[0024] The invention also relates to a method for obtaining the mutantof the present invention comprising:

[0025] (a) treating a Bacillus strain with a mutagen;

[0026] (b) growing the mutated Bacillus strain of step (a) undersuitable conditions for selecting the mutant; and

[0027] (c) selecting the mutant of step (b).

[0028] Substantially pure factor may be obtained from the mutant by:

[0029] (a) culturing a mutant of a Bacillus strain under suitableconditions;

[0030] (b) recovering a supernatant of the culture of the mutant of step(a) and

[0031] (c) isolating the factor from the supernatant of step (b) toobtain the substantially pure factor.

[0032] The factor obtained from said mutant may be formulated into acomposition comprising the factor and a pesticidal carrier as well asthe factor and a Bacillus related pesticide, chemical pesticide and/or avirus with pesticidal properties. These compositions may be used forcontrolling a pest, decreasing the resistance of a pest to a Bacillusrelated pesticide comprising exposing the pest to a compositioncomprising the factor and a pesticidally acceptable carrier, orpotentiating the pesticidal activity of a Bacillus related pesticide.

5. BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 schematically shows the general procedure used forpurifying Ia.

[0034]FIG. 2 shows the ¹³C NMR spectrum of Ia.

[0035]FIG. 3 shows the proton NMR spectrum of Ia.

[0036]FIG. 4 shows the results of nOe experiments on the acetylatedderivative of Ia.

6. DETAILED DESCRIPTION OF THE INVENTION

[0037] The parental Bacillus strain may be, e.g., Bacillus subtilis,Bacillus licheniformis, or Bacillus thuringiensis. The parental Bacillusthuringiensis may be a wild-type strain which includes but is notlimited to Bacillus thuringiensis subsp. kurstaki, Bacillusthuringiensis subsp. aizawai, Bacillus thuringiensis subsp. galleriae,Bacillus thuringiensis subsp. entomocidus, Bacillus thuringiensis subsp.tenebrionis, Bacillus thuringiensis subsp. thuringiensis, Bacillusthuringiensis subsp. alesti, Bacillus thuringiensis subsp. canadiensis,Bacillus thuringiensis subsp. darmstadiensis, Bacillus thuringiensissubsp. dendrolimus, Bacillus thuringiensis subsp. finitimus, Bacillusthuringiensis subsp. kenyae, Bacillus thuringiensis subsp. morrisoni,Bacillus thuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp.toumanoffi, Bacillus thuringiensis subsp. toumanoffi, Bacillusthuringiensis subsp. pondicheriensis, Bacillus thuringiensis subsp.shandogiensis, Bacillus thuringiensis subsp. sotto, Bacillusthuringiensis subsp. nigeriae, Bacillus thuringiensis subsp.yunnanensis, Bacillus thuringiensis subsp. dakota, Bacillusthuringiensis subsp. nidiana, Bacillus thuringiensis subsp. tohokuensis,Bacillus thuringiensis subsp. kumamotoensis, Bacillus thuringiensissubsp. tochigiensis, Bacillus thuringiensis subsp. thompsoni, Bacillusthuringiensis subsp. wuhanensis, Bacillus thuringiensis subsp.kyushuensis, Bacillus thuringiensis subsp. ostriniae, Bacillusthuringiensis subsp. tolworthi, Bacillus thuringiensis subsp. pakistani,Bacillus thuringiensis subsp. japonensis, Bacillus thuringiensis subsp.colmeri, Bacillus thuringiensis subsp. pondicheriensis, Bacillusthuringiensis subsp. shandongiensis, Bacillus thuringiensis subsp.neoleonensis, Bacillus thuringiensis subsp. coreanensis, Bacillusthuringiensis subsp. silo, Bacillus thuringiensis subsp. mexcanensis,and Bacillus thuringiensis subsp. israelensis.

[0038] In a specific embodiment, the parental Bacillus thuringiensisstrain is Bacillus thuringiensis subsp. kurstaki. In a most specificembodiment, the parental strain is Bacillus thuringiensis subsp.kurstaki strain EMCC0086 (deposited with the NRRL as NRRL B-21147). Themutant in yet another specific embodiment has the identifyingcharacteristics of EMCC0129, deposited with the NRRL and having theaccession number NRRL B-21445, or has the identifiying characteristicsof EMCC0130, deposited with the NRRL and having the accession numberNRRL B-21444.

[0039] 6.1. Methods of Obtaining the Mutant

[0040] The parental strain may be treated with a mutagen to induce amutational event. Specifically, in one method of mutating Bacillusthuringiensis strains and selecting such mutants that are capable ofproducing substantially larger amounts of factor than their parentalstrains, the parental strain is:

[0041] i) treated with a mutagen,

[0042] ii) the treated cells are cultured in a suitable culture medium(e.g., NSMP medium); and

[0043] iii) cells are selected which produce a larger amount of factor.

[0044] In step (i), the mutagen for example may be a chemical mutagenN-methyl-N′-nitro-N-nitrosoguanidine, N,N′-dinitro-N′-nitrosoguanidine,or ethyl methanesulfonate, gamma irradiation, X-ray and/orUV-irradiation.

[0045] The cells are selected by assaying for the production of thefactor, for example, by capillary electrophoresis or immunoassay usingan antibody to the factor.

[0046] Another method of obtaining the high producing mutants of theinvention may be contemplated such as growing the parent strain in aliquid medium and selecting spontaneous mutants after spreading theculture broth on an agar medium suitable for selection of mutants.

[0047] Other methods of screening for the high producing mutants of theinvention may be contemplated such as separating the mutants from othermaterial on the basis of mass directly through centrifugation or othermeans of separating for mass.

[0048] 6.2. Obtaining the Factor

[0049] The Bacillus mutants of the present invention may be culturedusing media and fermentation techniques known in the art (see, forexample, Rogoff et al., 1969, J. Invertebrate Path. 14:122-129; Dulmageet al., 1971, J. Invertebrate Path. 18:353-358; Dulmage et al., inMicrobial Control of Pests and Plant Diseases, H. D. Burges, ed.,Academic Press, N.Y., 1980). In a specific embodiment, the fermentationmedia may comprise hydrolyzed protein, hydrolyzed carbohydrate, salts,and trace metal. The fermentation media may also optionally comprise oneor more amino acids. Upon completion of the fermentation cycle, thesupernatant can be recovered by separating B.t. spores and crystals fromthe fermentation broth by means well known in the art, e.g.,centrifugation and/or ultrafiltration. The factor is contained in thesupernatant which may be recovered by means well known in the art, e.g.,ultrafiltration, evaporation, and spray-drying. This procedure is morespecifically described in the sections which follow.

[0050] Purification of the factor can be carried out by variousprocedures known in the art, including but not limited to chromatography(e.g., ion exchange, affinity, and size exclusion columnchromatography), electrophoretic procedures, differential solubility,extraction, or any other standard technique known in the art.

[0051] The potentiating activity of the factor of the pesticidalactivity of Bacillus related pesticide, virus having pesticidalactivity, or chemical pesticide against various pests may be assayedusing procedures known in the art, such as an artificial insect dietincorporated, artificial diet overlay, leaf painting, leaf dip, andfoliar spray. Specific examples of such assays are given in Section 7,infra. The amount of factor produced may be quantitated, for example, bycapillary electrophoresis.

[0052] The factor may have a molecular weight of from about 350 to about1200 or in a specific embodiment from about 350 to about 700.

[0053] The factor potentiates the pesticidal activity of a Bacillusrelated pesticide at least about 1.5 fold to optionally about 1000 fold,preferably from about 100 fold to about 400 fold. In a specificembodiment, the factor potentiates the pesticidal activity of a Bacillusthuringiensis delta-endotoxin including but not limited to a CryI(including but not limited to CryIA, CryIB, and CryIC), CryII, CryIII,CryIV, CryV, or CryVI protein in full-length form or a proteolyticallyprocessed, truncated form, from about 1.5 fold to about 1000 fold. In amost specific embodiment, the factor potentiates a B.t. delta-endotoxinfrom about 100 fold to about 400 fold. The factor may also potentiatethe pesticidal activity of a chemical pesticide and/or a virus withpesticidal properties.

[0054] The factor may also be water soluble, stable in water up to about100° C. for at least about 5 minutes, stable when subjected to directsunlight for at least about 10 hours, and/or stable at a pH of about 2for about 10 days. The factor may have 13 carbons. Additionally, thefactor may have ¹H NMR shifts at about δ81.5, 3.22, 3.29, 3.35, 3.43,3.58, 3.73, 3.98, 4.07, 4.15, 4.25, 4.35 and ¹³C shifts at about 31.6,37.2, 51.1, 53.3, 54.0, 54.4, 61.5, 61.6, 64.1, 65.6, 158.3, 170.7, and171.3.

[0055] In a most specific embodiment said factor has the structure Ia orsalt thereof. The salt would be capable of potentiating a Bacillusrelated pesticide.

[0056] 6.3. Compositions Comprising the Factor

[0057] The factor obtained from the mutants of the present invention canbe formulated alone; with a Bacillus related pesticide, which asdefined, supra, is a Bacillus strain, spore, protein or fragment, orother substance, thereof, with activity against or which kills pests orprotects plants against a pest; with a chemical pesticide and/or anentomopathogenic virus and an acceptable carrier into a pesticidalcomposition(s), that is, for example, a suspension, a solution, anemulsion, a dusting powder, a dispersible granule, a wettable powder, anemulsifiable concentrate, an aerosol or impregnated granule. Examples ofsuch Bacillus strains include, but are not limited to, Bacillusthuringiensis subsp. kurstaki (marketed as DIPEL™ from AbbottLaboratories, Inc., JAVELIN™ from Sandoz, BIOBIT™ from Novo Nordisk A/S,FORAY™ from Novo Nordisk A/S, BIOCOT™ from Novo Nordisk A/S, MVP™ fromMycogen, BACTOSPEINE™ from Novo Nordisk A/S, and THURICIDE™ fromSandoz); Bacillus thuringiensis subsp. aizawai (marketed as FLORBAC™from Novo Nordisk A/S, and XENTARI™ from Abbots Laboratories, Inc.);Bacillus thuringiensis subsp. tenebrionis (marketed as NOVODOR™ fromNovo Nordisk A/S, TRIDENT™ from Sandoz, M-TRAK™ and M-ONE™ from Mycogen,and DITERRA™ from Abbott Laboratories Inc.); Bacillus thuringiensissubsp. israelensis (marketed as either BACTIMOS™ or SKEETAL™ from NovoNordisk A/S, TEKNAR™ from Sandoz, and VECTOBAC™ from AbbottLaboratories, Inc.); Bacillus thuringiensis kurstaki/tenebrionis(marketed as FOIL™ from Ecogen); Bacillus thuringiensis kurstaki/aizawai(marketed as CONDOR™ from Ecogen and AGREE™ from Ciba-Geigy); andBacillus thuringiensis kurstaki/kurstaki (marketed as CUTLASS™ fromEcogen). The Bacillus related protein may be selected from the groupincluding, but not limited to, CryI, CryII, CryIII, CryIV, CryV, andCryVI. The chemical pesticide may be, for example, an insect growthregulator such as diflubenzuron, a carbamate such as thiodicarb andmethomyl, an organophosphate such as chlorpyrifos, a pyrethroid such ascypermethrin and esfenvalerate, inorganic fluorine such as cryolite, anda pyrrole. The entomopathogenic virus may be a baculovirus, e.g.,Autographa californica nuclear polyhedrosis virus (NPV), Syngraphafalcifera NPV, Cydia pomonella GV (granulosis virus), Heliothis zea NPV,Lymantria dispar NPV, Orgyia pseudotsugata NPV, Spodoptera exigua NPV,Neodiprion lecontei NPV, Neodiprion sertifer NPV, Harrisina brilliansNPV, and Endopiza viteana Clemens NPV.

[0058] In compositions comprising the substance and a Bacillus relatedpesticide, the substance may be present in the amount of at least about0.1 g/BIU or 0.05 g factor per g Bacillus delta-endotoxin and spore,optionally to about 300 g/BIU or 150 g substance per g Bacillusdelta-endotoxin and spore, preferably 2 g/BIU or 1 g substance per gBacillus delta-endotoxin and spore. As defined herein “BIU” is billioninternational units as determined by bioassay. The bioassay compares thesample to a standard Bacillus reference material using Trichoplusia nior other pest as the standard test insect. The potency is determined bydividing the reference standard LC₅₀ then multiplying by the referencestandard potency.

[0059] In another embodiment, the composition may comprise the factor insubstantially pure form or a supernatant from Bacillus in dry,concentrated, or liquid form and a pesticidally acceptable carrier,examples of which are disclosed, infra. This composition may be appliedseparately to a plant, e.g., transgenic plants. Specifically, thecomposition may be applied to a plant previously containing andexpressing a Bacillus thuringiensis gene. In another embodiment, thecomposition may be applied to a plant previously exposed to a Bacillusthuringiensis composition. In another embodiment, the composition may beapplied to other environments of a dipteran pest(s), e.g., water orsoil. The substance is present in the composition at a concentration ofabout 0.001% to about 60% (w/w).

[0060] The composition comprising the substance and a pesticidallyacceptable carrier in addition to controlling a pest may also be used todecrease the resistance of a pest to a pesticide. Alternatively, thecomposition may be used to potentiate a Bacillus related pesticide. Thecomposition in one embodiment may be applied at the same time as thepesticide in an amount of at least about 2 g substance/BIU up tooptionally about 300 g substance/BIU. In another embodiment, thecomposition may be applied up to about 24 hours after the pesticide asan adjuvant to extend the efficacy of residual pesticide.

[0061] Such compositions disclosed above may be obtained by the additionof a surface active agent, an inert carrier, a preservative, ahumectant, a feeding stimulant, an attractant, an encapsulating agent, abinder, an emulsifier, a dye, a U.V. protectant, a buffer, a flow agent,or other component to facilitate product handling and application forparticular target pests.

[0062] Suitable surface-active agents include anionic compounds such asa carboxylate, for example, a metal carboxylate of a long chain fattyacid; a N-acylsarcosinate; mono or di-esters of phosphoric acid withfatty alcohol ethoxylates or salts of such esters; fatty alcoholsulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate orsodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylatedalkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkylaryl sulphonates such as alkyl-benzene sulphonates or loweralkylnaphthalene sulphonates, e.g., butyl-naphthalene sulphonate; saltsor sulphonated naphthalene-formaldehyde condensates; salts ofsulphonated phenol-formaldehyde condensates; or more complex sulphonatessuch as the amide sulphonates, e.g., the sulphonated condensationproduct of oleic acid and N-methyl taurine or the dialkylsulphosuccinates, e.g., the sodium sulphonate or dioctyl succinate.Non-ionic agents include condensation products of fatty acid esters,fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substitutedphenols with ethylene oxide, fatty esters of polyhydric alcohol ethers,e.g., sorbitan fatty acid esters, condensation products of such esterswith ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters,block copolymers of ethylene oxide and propylene oxide, acetylenicglycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylatedacetylenic glycols. Examples of a cationic surface-active agent include,for instance, an aliphatic mono-, di-, or polyamine as an acetate,naphthenate or oleate; an oxygen-containing amine such as an amine oxideof polyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

[0063] Examples of inert materials include inorganic minerals such askaolin, mica, gypsum, fertilizer, phyllosilicates, carbonates, sulfates,or phosphates; organic materials such as sugar, starches, orcyclodextrins; or botanical materials such as wood products, cork,powdered corncobs, rice hulls, peanut hulls, and walnut shells.

[0064] The compositions of the present invention can be in a suitableform for direct application or as a concentrate or primary compositionwhich requires dilution with a suitable quantity of water or otherdiluent before application. The pesticidal concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly. The compositioncontains 1 to 98% of a solid or liquid inert carrier, and 0 to 50%,preferably 0.1 to 50% of a surfactant. These compositions will beadministered at the labeled rate for the commercial product, preferablyabout 0.01 pound to 5.0 pounds per acre when in dry form and at about0.01 pint to 25 pints per acre when in liquid form.

[0065] In a further embodiment, the Bacillus thuringiensis crystaldelta-endotoxin and/or factor can be treated prior to formulation toprolong the pesticidal activity when applied to the environment of atarget pest as long as the pretreatment is not deleterious to thecrystal delta-endotoxin or substance. Such treatment can be by chemicaland/or physical means as long as the treatment does not deleteriouslyaffect the properties of the composition(s). Examples of chemicalreagents include, but are not limited to, halogenating agents; aldehydessuch as formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropranol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason, pi Animal Tissue Techniques, W. H. Freemanand Co., 1967).

[0066] The compositions of the invention can be applied directly to theplant by, for example, spraying or dusting at the time when the pest hasbegun to appear on the plant or before the appearance of pests as aprotective measure. Plants to be protected within the scope of thepresent invention include, but are not limited to, cereals (wheat,barley, rye, oats, rice, sorghum and related crops), beets (sugar beetand fodder beet), drupes, pomes and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries, andblackberries), leguminous plants (alfalfa, beans, lentils, peas,soybeans), oil plants (rape, mustard, poppy, olives, sunflowers,coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants(cucumber, marrows, melons), fibre plants (cotton, flax, hemp, jute),citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables(spinach, lettuce, asparagus, cabbages and other brassicae, carrots,onions, tomatoes, potatoes), lauraceae (avocados, cinnamon, camphor),deciduous trees and conifers (linden-trees, yew-trees, oak-trees,alders, poplars, birch-trees, firs, larches, pines), or plants such asmaize, turf plants,. tobacco, nuts, coffee, sugar cane, tea, vines,hops, bananas and natural rubber plants, as well as ornamentals. Thecomposition can be applied by foliar, furrow, broadcast granule,“lay-by”, or soil drench application. It is generally important toobtain good control of pests in the early stages of plant growth as thisis the time when the plant can be most severely damaged. The spray ordust can conveniently contain another pesticide if this is thoughtnecessary. In a preferred embodiment, the composition of the inventionis applied directly to the plant.

[0067] The compositions of the present invention can also be applieddirectly to ponds, lakes, streams, rivers, still water, and other areassubject to infestation by dipteran pests, especially pests of concern topublic health. The composition can be applied by spraying, dusting,springling, or the like.

[0068] The compositions of the present invention may be effectiveagainst insect pests of the order Lepidoptera, e.g., Achroia grisella,Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon,Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagastakuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi,Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara,Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneurasp., Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydialatiferreanus, Cydia pomonella, Datana integerrima, Dendrolimussibericus, Desmia funeralis, Diaphania hyalinata, Diaphania nitidalis,Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria,Eoreuma loftini, Ephestia elutella, Erannis tiliaria, Estigmene acrea,Eulia salubricola, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileucaoliviae, Homoeosoma electellum, Hyphantria cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis,Lymantria dispar, Macalla thyrsisalis, Malacosoma sp., Mamestrabrassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta,Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota sultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tineola bisselliella, Trichoplusia ni, Udearubigalis, Xylomyges curialis, Yponomeuta padella; order Diptera, e.g.,Aedes sp., Andes vittatus, Anastrepha ludens, Anastrepha suspensa,Anopheles barberi, Anopheles quadrimaculatus, Armigeres subalbatus,Calliphora stygian, Calliphora vicina, Ceratitis capitata, Chironomustentans, Chrysomya rufifacies, Cochliomyia macellaria, Culex sp.,Culiseta inornata, Dacus oleae, Delia antiqua, Delia platura, Deliaradicum, Drosophila melanogaster, Eupeodes corollae, Glossina austeni,Glossina brevipalpis, Glossina fuscipes, Glossina morsitans centralis,Glossina morsitans morsitans, Glossina moristans submorsitans, Glossinapallidipes, Glossina palpalis gambiensis, Glossina palpalis palpalis,Glossina tachinoides, Haemagogus equinus, Haematobia irritans, Hypodermabovis, Hypoderma lineatum, Leucopis ninae, Lucilia cuprina, Luciliasericata, Lutzomyia longlpaipis, Lutzomyia shannoni, Lycoriella mali,Mayetiola destructor, Musca autumnalis, Musca domestica, Neobellieriasp., Nephrotoma suturalis, Ophyra aenescens, Phaenicia sericata,Phlebotomus sp., Phormia regina, Sabethes cyaneus, Sarcophaga bullata,Scatophaga stercoraria, Stomoxys calcitrans, Toxorhynchites amboinensis,Tripteroides bambusa. However, the compositions of the invention mayalso be effective against insect pests of the order Coleoptera, e.g.,Leptinotarsa sp., Acanthoscelides obtectus, Callosobruchus chinensis,Epilachna varivestis, Pyrrhalta luteola, Cylas formicarius elegantulus,Listronotus oregonensis, Sitophilus sp., Cyclocephala borealis,Cyclocephala immaculata, Macrodactylus subspinosus, Popillia japonica,Rhizotrogu-s majalis, Alphitobius diaperinus, Palorus ratzeburgi,Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum, Triboliumconfusum, Tribolius destructor; Acari, e.g., Oligonychus pratensis,Panonychus ulmi, Tetranychus urticae; Hymenoptera, e.g., Iridomyrmexhumilis, Solenopsis invicta; Isoptera, e.g., Reticulitermes hesperus,Reticulitermes flavipes, Coptotermes formosanus, Zootermopsisangusticollis, Neotermes connexus, Incisitermes minor, Incisitermesimmigrans; Siphonaptera, e.g., Ceratophyllus gallinae, Ceratophyllusniger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalides canis,Ctenocephalides felis, Echicnophaga gallinacea, Pulex irritans,Xenopsylla cheopis, Xenopsylla vexabilis, Tunga penetrans; andTylenchida, e.g., Melodidogyne incognita, Pratylenchus penetrans.

[0069] The following examples are presented by way of illustration, notby way of limitation.

7. EXAMPLE: Characterization of Ia

[0070] As detailed herein, Ia is recovered and purified. Thecharacterization of Ia is detailed infra.

[0071] 7.1. Recovery and Purification of IA

[0072]B. thuringiensis subsp. kurstaki strain EMCC0086 (deposited withthe NRRL as B-21147) is fermented for 72 hours at 30° C. in a mediumcomprised of a carbon source such as starch, hydrolyzed starch, orglucose and a nitrogen source such as protein, hydrolyzed protein, orcorn steep liquor. The production of Ia is detected at 13 hours into thefermentation. Peak activity is found to be at approximately 30 hours.

[0073] Supernatant from a B. thuringiensis subsp. kurstaki fermentationis recovered by centrifugation and then is clarified by ultrafiltrationthrough a 30 kDa MW-CO membrane using a Rhone Poulenc UF system. The 30kDa filtration removed any remaining cell debris, crystaldelta-endotoxin, spores, and soluble protein greater than 30 kDamolecular mass. The permeate is concentrated 10 fold by evaporation. Thepermeate is centrifuged and then 0.2μ filtered to further removeinsolubles from the broth, leaving a clear broth containing Ia.

[0074] The purification of Ia to homogeneity is achieved using amulti-step purification procedure shown schematically in FIG. 1. Inconjunction with the recovery protocol outlined above, the purificationproceeded with a 5 kDa ultrafiltration seep. The permeate from the 5 kDaultrafiltration is adsorbed to a Sulfopropyl (SP) cation exchange resinand eluted with an ammonium acetate solution. The compound is thenconcentrated approximately 30x by lyophilization, and the salt and othercontaminants are removed with a BioRad P2 size exclusion column. Thepool from the P2 column is run over a high resolution SP HPLC cationexchange column which yielded a homogeneous compound. The contaminatingsalt is removed by repeated lyophilization.

[0075] Activity is monitored by a Spodoptera exigua micro-bioassay, andpurity is determined by capillary electrophoresis. Sample consisting of50 μl of Ia and 50 μl of CryIA(c) protein (15 μg/ml) purified fromBIOBIT™ FC (100 μl), is applied to individual wells of a jelly traycontaining 500 μl of solidified artificial insect diet. The trayscontaining the various samples are air dried. Two to four 2nd or early3rd instar Spodoptera exigua are added to the wells containing the driedsample. The wells are sealed with mylar poked with holes and areincubated for 2-3 days at 30° C. Degree of stunting and percentmortality are then recorded. Typically, 5 replicate wells are run foreach sample.

[0076] 7.2. Structure Eludication

[0077] The active compound is found to be water soluble but is notsoluble in organic solvents. It is positively charged and reacted withninhydrin as evidenced by silica thin layer chromatography. ¹³C andproton NMR of the compound are shown in FIGS. 2 and 3, respectively. ¹³CNMR experiments revealed the presence of 13 carbons (referenced to3-[trimethylsilyl propionic acid). A DEPT experiment determined thatthere are three quaternary carbons (C), seven methines (CH), threemethylenes (CH₂) and no methyl groups (CH₃). Using proton couplingexperiments such as 1-D decoupling and COSY, one large spin systemcontaining eight carbons is identified. In addition, a smaller spinsystem consisting of two carbons is present. A carbon proton correlationexperiment (HMBC) enabled assignment of each proton resonance in themolecule to its attached carbon.

[0078] Treatment of the active compound (13 mg) with acetic anhydride inpyridine resulted in the formation of an acetylated derivative which ismuch less polar. This derivative is purified by HPLC to give 3 mg ofpure acetylated derivative. Mass spectroscopy analysis revealed that thederivative has 7 acetates and a molecular weight of 690, which gives amolecular weight of 396 for the active compound and indicates that aneven number of nitrogens are present. Also, fragments containing 6acetates and 5 acetates are detected. High resolution data for 5 and 6acetate daughter ions are 645.2594 (6 acetates) and 607.2519 (5acetates) which indicate the following molecular formula for Ia,C₁₃H₂₈N₆O₈.

[0079] Treatment of the active compound (13 mg) with 6 N HCl gave aderivative which is ninhydrin positive. These results indicate thepresence of amide bonds. The derivative had the same R_(f) value asdetermined by thin layer chromatography as 2,3-diaminopropionic acid.These results along with NMR data, suggest the presence of 2,3diaminopropionic acid.

[0080] Another technique used to analyzed Ia is nOe (Nuclear OverhauserEffect) which can detect proximity of protons to one another throughspace. nOe is carried out on an acetylated derivative of Ia. In a twodimensional nOe experiment (NOESY), nOes are observed between an N-Hproton at 8.06 ppm and the 5.17 proton (FIG. 4).

[0081] The following structure has been elucidated for Ia

[0082] It can be classified as a ureido amide. Constituents include 2amides, a urea, two aminos, and five hydroxyls. It contains seven chiralcenters.

[0083] 7.3. Properties of Ia

[0084] The isolated Ia is found to potentiate the activity of Bacillusthuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawaicrystal delta-endotoxin pesticidal proteins toward Spodoptera exiguaregardless of the form of the pesticidal proteins. The pesticidalactivity of formulated B.t.k., isolated crystals, full-length (130 kDamolecular mass) or truncated CryIA proteins (˜65 kDa molecular mass) areall potentiated. The activity of CryII and CryIC inclusions are alsopotentiated. It is also found to potentiate the activity of theindividual truncated CryIA(a), (b), and (c) proteins. Incubation time ofIa with the Cry protein is not found to be critical for bioactivity.However, Ia is inactive alone. The level of potentiation is found to be100-200 fold for the truncated CryIA proteins, CryII and CryICinclusions and approximately 320 fold with full-length CryIA(c) (seeTables I and II respectively). Specifically, for full-length protein,0.75 μg/ml CryIA(c) produced the same insect mortality/stunt score whenIa is included as 240 μg/ml of CryIA(c) alone. In the case of thetruncated CryIA(c), an OD₂₈₀ of 0.0006 gave the same stunt score incombination with Ia as the same sample of CryIA(c) tested alone with anOD₂₈₀ of 0.075. CryII inclusions, at a concentration of 0.6 μg/ml gavethe same stunt score and similar mortality in combination with Ia asCryII protein alone at 75 μg/ml, a 125 fold potentiation. CryICinclusions, at 0.3 μg/ml with the addition of Ia gave similar mortalityand stunt score as 75 μg/ml of the CryIC protein alone, which reflects a250 fold level of potentiation. The concentration of CryIA protein thatproduced stunting yielded mortality on addition of Ia.

[0085] Ia is found to be stable upon boiling for 5 minutes, but losesall activity upon autoclaving (>190C). Further, it is stable whensubjected to direct sunlight for at least 10 hours. Ia is stable at pH 2for 3 days, but unstable at pH 12. It is found to lose all activity whenexposed to periodic acid or concentrated HCl. TABLE I POTENTIATIONEFFECTS OF Ia WITH PURIFIED TRUNCATED Bt PROTEIN Bt Protein SpodopteraExigua Type OD280 Ia Mortality* Stunt Score† CryIa(a) 0.055 − 0/5 2.20.040 − 0/5 2.2 0.020 − 0/5 2.0 0.020 + 2/5 0.0 0.010 + 0/5 0.2 0.005 +0/5 0.0 0.0025 + 0/5 0.4 0.0012 + 0/5 1.8 0.0006 + 0/5 1.6 CryIA(c)0.075 − 0/5 3.4 0.040 − 0/5 2.6 0.020 − 0/5 2.8 0.020 + 1/5 0.0 0.010 +0/5 0.2 0.005 + 1/5 0.0 0.0025 + 2/5 2.0 0.0012 + 0/5 1.0 0.0006 + 1/51.0 None NA + 0/5 4.0 None NA − 0/5 4.0

[0086] TABLE II POTENTIATION EFFECTS OF Ia WITH Bt PROTEIN Bt ProteinSpodoptera Exigua Type μg/ml Ia Mortality* Stunt Score† CryIA(c) 240 −1/5 0.5 120 − 0/5 2.2 60 − 0/5 2.2 30 − 0/5 4.0 60 + 5/5 — 30 + 5/5 —15 + 4/5 0.0 3 + 4/5 1.0 0.8 + 2/5 1.6 CryII 300 − 1/5 0.8 150 − 2/5 0.775 − 1/5 0.2 38 − 0/5 0.8 19 − 0/5 1.6 9 − 0/5 1.8 5 − 1/5 4.0 38 + 3/51.0 19 + 2/5 0.5 9 + 3/5 0.0 5 + 1/5 0.5 2.4 + 1/5 0.0 1.2 + 3/5 0.50.6 + 2/5 0.3 CryII 300 − 2/5 0.3 150 − 2/5 0.0 75 − 1/5 0.8 38 − 0/53.2 38 + 5/5 — 19 + 5/5 — 9 + 5/5 — 5 + 4/5 0.0 2.4 + 1/5 0.0 1.2 + 5/5— 0.6 + 3/5 1.5 0.3 + 2/5 1.3 None NA − 0/5 4.0 None NA + 0/5 4.0

[0087] 7.4. Evaluation of Other Subspecies of Bacillus thuringiensis andOther Species of Bacilli

[0088] Several Bacillus species are evaluated for production of Ia. Thestrains are fermented for 72 hours at 30° C. in a medium comprised of acarbon source such as starch, hydrolyzed starch, or glucose and anitrogen source such protein, hydrolyzed protein, or corn steep liquor.The supernatants are tested for Ia production using the Spodopteraexigua micro-bioassay described supra. B. thuringiensis subsp. aizawaistrain EMCC0087 (deposited with the NRRL as NRRL B-21148) and B.thuringiensis subsp. galleriae (deposited with the NRRL) are found toproduce Ia in about the same concentration as B. thuringiensis subsp.kurstaki.

[0089] Ia is also produced in B. subtilis, B. cereus, B.t. subsp.alesti, B.t. subsp. canadiensis, B.t. subsp. darmstadiensis, B.t. subsp.dendrolimus, B.t. subsp. entomocidus, B.t. subsp. finitimus, B.t. subsp.israelensis, B.t. subsp. kenyae, B.t. subsp. morrisoni, B.t. subsp.subtoxicus, B.t. subsp. tenebrionis, B.t. subsp. thuringiensis, and B.t.subsp. toumanoffi, B. cereus, B. subtilis, and B. thuringiensis subsp.kurstaki cry- spo- mutant as determined by capillary electrophoresis.

[0090] Specifically, a Beckman P/ACE Capillary Electrophoresis Systemequipped with a 50μm×57 cm uncoated capillary, 0.2 M phosphate pH 6.8buffer, voltage at 15KV, and detection at 200 nm is used for quantifyingthe level of Ia. Sample volumes are 20 nl with a run time of 25 minutes.

[0091] A standard curve is generated using purified Ia as the standardat levels of 1.25 mg/ml, 0.625 mg/ml, 0.3125 mg/ml, 0.156 mg/ml, and0.078 mg/ml. A linear calibration curve is generated. The resultanty=mx+b equation is used to generate the concentration of Ia in eachsample.

[0092] Before each run, the capillary is flushed with running buffer(0.2 M phosphate, pH 6.8) for three minutes. After each 25 minute run,the capillary is flushed with 1 N NaOH for 1 minute, filtered HPLC waterfor 1 minute, 0.5 M phosphoric acid for 3 minutes, and filter HPLC waterfor 1 minute. The area under each peak is integrated and the peak areais determined and a final concentration is calculated from the standardcurve.

[0093] 7.5. Evaluation of B.t. Products

[0094] The amount of Ia present in various commercially available B.t.products is determined by capillary electrophoresis described in Section6.4, supra. BACTOSPEINE™, JAVELIN™, NOVODOR™, SPHERIMOS™, BACTIMOS™,FORAY™, FLORBAC™ and BIOBIT™ are obtained from Novo Nordisk A/S.XENTARI™ and DIPEL™ are obtained from Abbott Laboratories. AGREE™ isobtained from Ciba-Geigy; MVP™ is obtained from Mycogen and CUTLASS™ isobtained from Ecogen.

[0095] The results are shown in Table III, infra and indicate that Ia ispresent in varying quantities ranging from less than 0.001 g Ia/BIU to0.071 g Ia/BIU. TABLE III Ia IN Bacillus thuringiensis PRODUCTS PRODUCTtype Lot Number Potency Ia g/BIU JAVELIN ™ WG Btk 9942281 32000 .071IU/mg XENTARI ™ Bta 58715PG 15000 .06 IU/mg AGREE ™ Bta/Btk RA20800425000 .033 IU/mg BIOBIT ™ HPWP Btk 5012 48950 .018 U/mgPIA BIOBIT ™ FCBtk AG46669071 8 .013 BIU/L FORAY ™ 48B Btk BBN7018 12.6 .012 BIU/LDIPEL ™ Btk 58739PG 32,000 .011 IU/mg FORAY ™ 76B Btk 20.0 .007 BIU/LBACTOSPEINE ™ Btk B0B001 123653 .003 IU/mg BACTOSPEINE ™ Btk KX02A100,000 .003 IU/mg BACTOSPEINE ™ Btk WP 16,000 <.001 IU/mg NOVODOR ™ Btt9024 16.3 9.5 × l0−9 Million g/LTU LTU/qt FLORBAC ™ Bta 082-31-1 30,000<.001 U/mg E SPHERIMOS ™ B. sphr BSN006 none MVP ™ Btk 21193542 noneCUTLASS ™ Btk/Btk none BACTIMOS ™ Bti BIB0024 11,700 none IU/mg

[0096] 7.6. Diet Incorporation Bioassays

[0097] B.t.k. activity is determined by an artificial diet incorporationbioassay using third instar Spodoptera exigua larvae, second instarHelicoverpa zea larvae, third instar Spodoptera frugiperda larvae,second instar Heliothis virescens larvae, third instar Trichoplusia nilarvae, third instar Pseudoplusia includens larvae, third instarPlutella xylostella larvae, third instar Spodoptera littoralis, andthird instar Mamestra brassicae larvae.

[0098] To determine the level of potentiation by adding Ia to B.t.products, ard establish the range of insects that are affected, dietincorporation bioassays are performed. In the experiments with highconcentrations of Ia against Spodoptera exigua (7.4-23.7 g Ia/BIU),purified Ia (70% active ingredient, 30% acetate counter ion) is used topotentiate BIOBIT™ FC (FC represents flowable concentrate). Theremaining data presented in Table IV shows the potentiation of BIOBIT™HPWP (high potency wettable powder) with Ia (0.658% active ingredient).S. littoralis and M. brassicae are tested using FLORBAC™ HPWP and Ia.

[0099] The various B.t. products are weighed and Ia is added to give 0.1to 237 g Ia/BIU. The volume is adjusted with 0.1% Tween™. The samplesare sonicated for 1 minute and then diluted to final volume. Neatsamples (without Ia) and reference substances are prepared as well.Reference substances include B.t.k. HD-1-S-1980 (obtained from the NRRL)which is assigned a potency of 16,000 international units (IU) permilligram and JAVELIN™ WG which has been assigned a potency of 53,000Spodoptera Units/mg (SU).

[0100] Standard artificial diet composed of water, agar, sugar, casein,wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts,and vitamins are prepared in a 20 L heated kettle. This provides enoughdiet to test 10 to 12 samples with seven different concentrations ofeach test substance. The B.t. solutions are serially diluted to give 16ml aliquots. Each aliquot is added to 184 g of molten diet. The mixtureis subsequently homogenized and then poured into a plastic tray bearing40 individual cells. Three control trays are prepared for each batch ofdiet. Once the diet has cooled and solidified, one insect of a known age(2-3 instar) is added to each cell, and the trays are covered with aperforated sheet of clear mylar. The trays are placed on racks andincubated for four days at 28° C. and 65% relative humidity.

[0101] After four days, insect mortality is rated. Each tray is given asharp blow against a table top, and larvae that did not move are countedas dead. Percent mortality is calculated and the data is analyzed viaparallel probit analysis. LC₅₀s, LC₉₀s, the slope of the regressionlines, coefficient of variation, and potencies are estimated. Samplesare run a minimum of 3 times or until three potencies are within 20% ofa calculated mean for each sample. To calculate the increase in activityassociated with each concentration of Ia, the LC₅₀ of the B.t./Ia sampleis corrected to reflect the amount of B.t. in the sample. The LC₅₀s ofthe paired neat samples are divided by the corrected LC₅₀ values to givethe fold reduction in LC₅₀ associated with Ia.

[0102] The following procedure is used to assay for Lobesia bothrana.Vine grapes attacked by Lobesia bothrana are collected in an unsprayedfield and larva is removed. A dilution series of Ia (250 μg/ml, 500μg/ml, and 1000 μg/ml) is made in water. One larva is put in the middleof the petri dish. If the larva is observed to drink, it is moved into apetri dish with freshly cut grape berries. The larvae are stored at 22°C. for 3-4 days.

[0103] As shown in Table IV, significant reductions in LC₅₀s areobserved for all species. TABLE IV Diet Incorporation Bioassays Increasein activity Insect g Ia per BIU Fold reduction in LC₅₀ Spodoptera exigua0.1 1.5 (BIOBIT ™ HPWP) 0.2 1.7 2.0 4.3 4.0 7.5 Spodoptera exigua 7.4 13(BIOBIT ™ FC) 15 26 30 34 118 59 237 79 Spodoptera frugiperda 0.2 2.2(BIOBIT ™ HPWP) 0.8 3.9 2.0 7.2 4.0 11.6 Trichoplusia ni 0.1 1.1(BIOBIT ™ HPWP) 0.2 1.2 2.0 2.0 4.0 3.1 Pseudoplusia includens 0.1 0(BIOBIT ™ HPWP) 0.2 1.2 0.8 2.1 2.0 2.4 4.0 3.4 Plutella xylostella 0.21.6 (BIOBIT ™ HPWP) 0.8 1.3 2.0 1.4 4.0 1.9 Helicoverpa zea 3.2 12.6(BIOBIT ™ HPWP) Heliothis virescens 3.2 4.2 (BIOBIT ™ HPWP) Lobesiabothrana 2.0 3.0 (BIOBIT ™ HPWP) Spodoptera littoralis 2.0 8.6(FLORBAC ™ HPWP) Mamestra brassicae 2.0 4.9 (FLORBAC ™ HPWP)

[0104] The potentiation of various products on Spodoptera exigua by Iais determined using diet incorporation bioassays described supra.Amounts of Ia added/BIU product are shown in Table V, infra. Ia/B.t.product mixture is incorporated into an agar-based wheat germ caseindiet. The insects are placed on the diet for four days and held at 28°C. Mortality is recorded and analyzed using probit analysis. LC₅₀, LC₉₀and potency are calculated from matched product lacking Ia. The resultsshown in Table V indicate that Ia potentiate various B.t.k. and B.t.a.products obtained from various sources. The B.t. strains contained inthese products are described in Section 5.2., supra. TABLE VPotentiation of B.t. Products on Spodoptera exigua Increase in activityProduct g Ia per BIU Fold reduction in LC₅₀ BACTOSPEINE ™ WP 0.4 1.041.7 2.3 CONDOR ™ 0.4 2.4 1.7 5.1 AGREE ™ 0.4 1.1 1.7 1.6 CUTLASS ™ 0.41.1 1.7 2.5 MVP ™ 0.4 6.0 1.7 7.7 2.0 12.1 FLORBAC ™ HPWP 0.2 1.1 0.82.0 DIPEL ™ 2X 0.2 1.2 0.8 2.3 2.0 3.9 JAVELIN ™ WG 0.2 0 0.8 1.08 2.02.9 XENTARI ™ 0.2 1.2 0.8 1.6 2.0 2.4

[0105] 7.7. Foliar Bioassays

[0106] Foliar bioassays are performed with second instar Spodopteraexigua larvae on broccoli plants using BIOBIT™ FC and Ia. The ratio ofIa to BIOBIT™ FC is the same 2 g Ia/BIU BIOBIT™ FC. The treatments areapplied to broccoli plants via a track sprayer in a carrier volume of 20gallons per acre. Leaves are excised from the plants after the spraydeposit had dried, and infested with second instar Spodoptera exigualarvae. The results are shown in Table VI, infra. 100% mortality isobserved at a rate of 8.7 BIU/hectare BIOBIT™ FC+Ia, while BIOBIT™ FCalone killed 92% of the larvae at 58.8 BIU/hectare and 8% at 17.6BIU/hectare. Treated plants are also placed in direct sunlight for eighthours, after which leaves are excised and infested. After eight hours insunlight, BIOBIT™ FC alone at 58.8 BIU/hectare gave 27% mortality, whileBIOBIT™ FC+Ia gave 100% mortality at 8.7 BIU/hectare.

[0107] A foliar assay done with early fourth instar larvae had BIOBIT™FC alone with 75% mortality at 52 BIU/hectare, and BIOBIT™ FC (FC isflowable concentrate) +Ia gave 100% mortality at 13 BIU/hectare. TABLEVI Foliar Bioassays Treatment BIU/hectare % mortality larval instarBIOBIT ™ FC 58.8 92% 2 BIOBIT ™ FC 17.6  8% 2 BIOBIT ™ FC + Ia 8.7 100% 2 BIOBIT ™ FC + 58.8 27% 2 8 hr sunlight BIOBIT ™ FC + Ia + 8.7 100%  28 hr sunlight BIOBIT ™ FC 52 75% 4 BIOBIT ™ FC + Ia 13 100%  4

[0108] 7.8. Field Trials

[0109] Field trials on garbonzo beans (Spodoptera exigua) demonstratedthat BIOBIT™ FC alone at 70 BIU/hectare gave 51% control while 2 gIa/BIU BIOBIT™ FC at 40 BIU/hectare provided 89% control (relative to notreatment). JAVELIN™ WG at 45 BIU/hectare gave 51% control.

[0110] Field trials on sweet corn (Spodoptera frugiperda) demonstratedthat at 39.5 BIU/hectare, 2 g Ia/BIU BIOBIT™ FC provided 84% control.

[0111] 7.9. Resistance Ratios

[0112] Colonies of susceptible and resistant Plutella xylostella arebioassayed. Resistant moths are field collected samples from Floridathat have developed B.t. resistance following intensive exposure toJAVELIN™ WG. BIOBIT™ HPWP with Ia is analyzed using a leaf-dip bioassay.Resistance to JAVELIN™ and XENTARI™ is assayed without Ia. Six cmdiameter cabbage leaf disks are dipped for 10 seconds into one of eightdifferent concentrations of B.t. products or B.t./Ia formulations.Concentrations range from 1 to 1000 ppm. The leaf disks are allowed toair dry for two hours and placed in plastic petri dishes with secondinstar (0.2 to 0.4 mg) larvae. Twenty five insects/dose/day arereplicated twice to give 50 insects/dose. After 72 hours at 27° C.,mortality is recorded. Dose mortality regression is analyzed with probitanalysis. Resistance ratios are calculated by dividing the LC₅₀ and LC₉₀values of the susceptible moths. The results are shown in Table VII andindicate that the BIOBIT™ HPWP potentiates with 2 g Ia/BIU and 4 gIa/BIU. Specifically, with 4 g Ia/BIU there is a 2 fold decrease in theLC₅₀ resistance ratio and a 10 fold decrease in the LC₉₀ resistanceratio. TABLE VII Plutella xylostella (B.t.k. Resistant) ResistanceRatios PRODUCT TESTED LC₅₀ RR LC₉₀ RR JAVELIN ™ WG 302.6 3829.7 BIOBIT ™HPWP 20.5 98.5 2.0 g Ia/BIU 23.2 88.0 BIOBIT ™ HPWP 4.0 g Ia/BIU 10.411.5 BIOBIT ™ HPWP XENTARI ™ 9.7 8.2

[0113] 7.10. Mutants

[0114] 7.10.1.Strain

[0115] The strains Bacillus thuringiensis subsp. kurstaki EMCC0086 (NRRLB-21147), Septoria nodorum (A04119), and Alterharia alternate (Strain 6,IM-SMP) are used.

[0116] 7.10.2. Funaal Growth Inhibition Assay

[0117] Mutants of the Bacillus thuringiensis subsp. kurstaki strainwhich produce the factor are identified using fungal agar plates ofSeptoria nodorum and Alternaria alternata. The factor inhibits growth ofboth fungi.

[0118] The two fungi are grown on PDA (potato dextrose agar) plates at26° C. for 10-14 days until sufficient sporulation is obtained.Alternaria alternata is incubated in alternating UV light and darkness(12 hrs. each). The spores are removed from the plates and suspended insterile water. Septoria nodorum spores are filtered through a sterile G1filter. The Alternaria alternata spore concentration is adjusted toapproximately 2×10⁵ per ml and the Septoria nodorum spore concentrationis adjusted to approximately 2×10⁶ per ml using a Burker-Turk orFuchs-Rosenthal counter. Samples of 1.5 ml of the spore suspension arethen mixed with 1.5 ml of sterile 40% glycerol in water, frozen for 1day at -20° C. and thereafter stored at −80° C. until use.

[0119] Test plates for the factor are prepared by mixing at 46° C. a 1.5ml spore sample (as described above) with 115 ml of antibiotic agarmedium No. 2 (25.5 g per liter) comprising streptomycin at a finalconcentration of 100 μg per ml, and transferring the mixture to sterileplastic trays (Nunc No. 101875). After the agar has solidified, mm holesare punched into the agar (120 holes per tray).

[0120] Samples of a culture are serially diluted in 100 μg ofstreptomycn per ml of sterile water and 10 μl of the diluted samples aredispensed into the trays prepared as described above. The trays areincubated at 26° C. for 2 days. Dilutions of the factor are also run asa positive control.

[0121] The sensitivity of the fungal growth inhibition assay is in therange of 0.05 gram of the factor per liter (barely visible) and 0.1 gramof the factor per liter (clear zone) for both fungi.

[0122] 7.10.3. Capillary zone Electrophoresis Measurement of Factor

[0123] Cells and other insolubles are removed from a whole culture brothsample by centrifugation and/or by filtration through a 0.2 μm Nylonmembrane filter prior to analysis. A Beckman P/ACE CapillaryElectrophoresis System equipped with a 50 μm×47 cm uncoated capillary,running buffer consisting of 0.1 M phosphate pH 6.6, voltage at 15 KV,and detection at 200 nm is used for quantification of the factor. Beforeeach run, the capillary is flushed with the running buffer for 2minutes. The volume of the sample loaded is 20 nl.

[0124] The run time for analysis is 12 minutes with the factor elutingat approximately 8.5 minutes. The concentration of the factor isdetermined relative to a standard curve of the pure compound.

[0125] Between each 12 minute run, the capillary is flushed sequentiallywith 1 N sodium hydroxide for 0.5 minute, 0.1 N sodium hydroxide for 0.5minute, HPLC water for 0.5 minute, and 1.5 M phosphoric acid for 0.5minute.

[0126] 7.10.4. Mutaaenesis of B. thurinaiensis subsp. kurstaki Strain

[0127] Spores of the Bacillus thuringiensis subsp. kurstaki NB75 strainare treated first with gamma-rays at 700 Krads. The irradiated sporesare serially diluted, spread onto TY agar plates, and incubated at 30°C. for 2 days. Eighty mutants from the treatment are purified by singlecolony streaking on TY agar plates and are then transferred to 500 mlshake flasks containing 100 ml of medium with the following composition:Corn Steep Liquor 15 g/liter Maltodextrin 40 g/liter Potato Protein 30g/liter KH₂PO₄ 1.77 g/liter K₂HPO₄ 4.53 g/liter

[0128] The shake flasks are incubated at 30° C., 250 rpm for 3 days.

[0129] Samples of 5 ml are taken daily from each of the shake flaskcultures and centrifuged to pellet the cells. The supernatants arediluted 2-10 times in streptomycin at a concentration of 0.1 mg per mlof deionized water and then are tested for antifungal activity asdescribed in Section 8.2. Culture samples of those mutants producing thegreatest inhibition of fungal growth are then analyzed for the amount ofthe factor by capillary zone electrophoresis as described in Section8.3. The highest producing mutant from the first mutation is NBB-76.

[0130] The first generation mutant NBB-76 is then subjected to a secondmutation using N,N′-dinitro-N′-nitrosoguanidine (NTG). Specifically, themutant strain is cultivated overnight at 30° C., 240 rpm in a 500 mlshake flask containing 100 ml of TY broth with the following compositionadjusted to pH 7.3 before autoclaving: Tryptone 20 g/liter Yeast extract5 g/liter FeCl₂—4H₂O 6 mg/liter MnCl₂—4H₂O 1 mg/liter MgSO₄—7H₂O 15mg/liter

[0131] The overnight culture is diluted 100 times into a new shake flaskcontaining TY medium and cultivated at 30° C., 240 rpm until the culturereaches logarithmic growth (approximately 4 hours). A sample of 10 ml ofthe culture is removed from the shake flask and then germfiltrated usinga 0.45 μm Nalgene filter unit. The cells on the filter are resuspendedin 10 ml of TM buffer containing 100 μg of NTG per ml. TM buffer iscomprised of 6.05 g of Tris and 5.08 g of maleic acid per liter ofdeionized water adjusted to pH 6 with sodium hydroxide. The suspendedcells are incubated at 37° C. for 30 minutes and then are connected to avacuum source to remove the NTG from the cells. The cells are washed twotimes with 20 ml of M9 buffer before they are resuspended in 10 ml of M9buffer, serially diluted, spread onto TY agar plates, and incubated at30° C. for 2 days. M9 buffer is comprised of 8.78 g of Na₂HPO₄-2H₂O, 3 gof KH₂PO₄, 4 g of NaCl, and 0.2 g of MgSO₄-7H₂O per liter of deionizedwater. Mutants are isolated and tested as described above. The highestproducing mutant obtained from the second round of mutation is EMCC0130.

[0132] The second generation mutant EMCC0130 is then subjected to athird mutation using NTG as described above. The highest producingmutant obtained from the third round of mutation is NBC-217.

[0133] The third generation mutant NBC-217 is then subjected. to afourth mutation using NTG as described above. The highest producingmutant obtained from the fourth round of mutation is EMCC0129.

[0134] 7.10.5. Factor Production by Mutants and Parent Strain

[0135] Mutants EMCC0130, NBC-217, and EMCC0129 and the parent strainBacillus thuringiensis subsp. kurstaki EMCC0086 are grown in 250 mlshake flasks containing 50 ml of medium comprised of the followingcomponents supplemented with trace metals at 0.2 ml per liter and thenadjusted to pH 7 with H₃PO₄ prior to sterilization: Hydrolyzed VegetableProtein 30 g/liter Hydrolyzed Starch 40 g/liter K₂HPO₄ 5 g/liter MgSO₄0.3 g/liter

[0136] The shake flask cultures are incubated at 30° C., 250 rpm for 3days.

[0137] Quantitative analysis of the factor produced by the mutants andthe parent strain is performed by capillary zone electrophoresis asdescribed in Section 8.3. Quantitative analysis indicates that mutantEMCC0129 produces approximately 0.9 g of the factor per liter of culturebroth after 3 days in shake flasks while the parent strain producesapproximately 0.15 g per liter. Mutant EMCC0129 produces approximately6-fold more factor than the parent strain.

[0138] Table VIII: Production of Factor by Mutants of Bacillusthuringiensis subsp. kurstaki Mutant Factor (g/liter) Parent 0.15EMCC0130 0.65 NBC-217 0.65 EMCC0129 0.75

[0139] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

[0140] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

[0141] 8. Deposit of Microorganisms

[0142] The following strains of Bacillus thuringiensis have beendeposited according to the Budapest Treaty in the Agricultural ResearchService Patent Culture Collection (NRRL), Northern Regional ResearchCenter, 1815 University Street, Peoria, Ill., 61604, U.S.A. StrainAccession Number Deposit Date EMCC0086 NRRL B-21147 October 6, 1993EMCC0129 NRRL B-21445 May 23, 1995 EMCC0130 NRRL B-21444 May 23, 1995

[0143] The strains have been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of eachdeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

What is claimed is:
 1. A mutant of a Bacillus strain which produces afactor which potentiates the pesticidal activity of a Bacillus relatedpesticide, wherein the amount of the factor produced by the mutant isgreater than the amount of the factor produced by a correspondingparental strain, wherein said Bacillus strain is selected from the groupconsisting of Bacillus licheniformis, Bacillus subtilus, and Bacillusthuringiensis.
 2. The mutant according to claim 1, wherein the mutantproduces at least about 2 times more factor than the correspondingparental strain.
 3. The mutant according to claim 1, wherein the factorhas ¹H NMR shifts at about δ1.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73,3.98, 4.07, 4.15, 4.25, and 4.35, and ¹³C shifts at about 31.6, 37.2,51.1, 53.3, 54.0, 54.4, 61.5, 61.6, 64.1, 65.6, 158.3, 170.7, and 171.3.4. The mutant according to claim 1, wherein the factor has the structureI or salt thereof


5. The mutant according to claim 1, wherein the Bacillus strain is aBacillus thuringiensis strain.
 6. The mutant according to claim 5,wherein the Bacillus thuringiensis strain is selected from the groupconsisting of strains of Bacillus thuringiensis subsp. aizawai, Bacillusthuringiensis subsp. alesti, Bacillus thuringiensis subsp. canadiensis,Bacillus thuringiensis subsp. colmeri, Bacillus thuringiensis subsp.coreanensis, Bacillus thuringiensis subsp. dakota, Bacillusthuringiensis subsp. darmstadiensis, Bacillus thuringiensis subsp.dendrolimus, Bacillus thuringiensis subsp. entomocidus, Bacillusthuringiensis subsp. finitimus, Bacillus thuringiensis subsp. galleriae,Bacillus thuringiensis subsp. indiana, Bacillus thuringiensis subsp.israelensis, Bacillus thuringiensis subsp. kenyae, Bacillusthuringiensis subsp. kumamotoensis, Bacillus thuringiensis subsp.kurstaki, Bacillus thuringiensis subsp. kyushuensis, Bacillusthuringiensis subsp. japonensis, Bacillus thuringiensis subsp.mexcanensis, Bacillus thuringiensis subsp. morrisoni, Bacillusthuringiensis subsp. neoleonensis, Bacillus thuringiensis subsp.nigeriae, Bacillus thuringiensis subsp. ostriniae, Bacillusthuringiensis subsp. pakistani, Bacillus thuringiensis subsp.pondicheriensis, Bacillus thuringiensis subsp. shandongiensis, Bacillusthuringiensis subsp. silo, Bacillus thuringiensis subsp. sotto, Bacillusthuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp.tenebrionis, Bacillus thuringiensis subsp. thompsoni, Bacillusthuringiensis subsp. tochigiensis, Bacillus thuringiensis subsp.tohokuensis, Bacillus thuringiensis subsp. tolworthi, Bacillusthuringiensis subsp. toumanoffi, Bacillus thuringiensis subsp.wuhanensis, and Bacillus thuringiensis subsp. yunnanensis.
 7. The mutantaccording to claim 5, wherein the Bacillus thuringiensis strain is aBacillus thuringiensis subsp. kurstaki strain.
 8. The mutant accordingto claim 1, wherein the mutant has the identifying characteristics ofEMCC0129, deposited with the NRRL, having an accession number of NRRLB-wwww; or has the identifying characteristics of EMCC0130, depositedwith the NRRL, having an accession number of NRRL B-xxxx.
 9. The mutantaccording to claim 1, wherein the Bacillus related pesticide comprises aBacillus thuringiensis delta-endotoxin or a pesticidally-active fragmentthereof.
 10. The mutant according to claim 9, wherein the Bacillusthuringiensis delta-endotoxin or the pesticidally-active fragmentthereof is selected from the group consisting of CryI, CryII, CryIII,CryIV, CryV, and CryVI.
 11. The mutant according to claim 10, whereinthe Bacillus thuringiensis delta-endotoxin or the pesticidally-activefragment thereof is a CryIA delta-endotoxin or a pesticidally-activefragment thereof.
 12. The mutant according to claim 10, wherein theBacillus thuringiensis delta-endotoxin or the pesticidally-activefragment thereof is a CryIC delta-endotoxin or a pesticidally-activefragment thereof.
 13. The mutant according to claim 1, wherein theBacillus related pesticide comprises a Bacillus thuringiensis spore. 14.The mutant according to claim 1, wherein the factor is obtained by (a)culturing the mutant of the Bacillus strain under suitable conditions;(b) recovering a supernatant of the culture of the mutant of step (a);and (c) isolating the factor from the supernatant of step (b).
 15. Themutant according to claim 14, wherein the factor is obtained from thesupernatant of the culture of a Bacillus thuringiensis strain.
 16. Amethod for obtaining the mutant of claim 1 comprising (a) treating aBacillus strain with a mutagen; (b) growing the mutated Bacillus strainof step (a) under suitable conditions for selecting the mutant; and (c)selecting the mutant of step (b).
 17. A mutant of a Bacillus strainobtained according to the method of claim 16.